J. Physiol. (1977), 266, pp. 91-101 With 4 text-ftgurem Printed in Great Britain
91
ON THE EFFECT OF CALCIUM ON THE FREQUENCY OI MINIATURE END-PLATE POTENTIALS AT THE FROG NEUROMUSCULAR JUNCTION
BY GARY MATTHEWS AND WARREN 0. WICKELGREN From the Department of Physiology, University of Colorado Medical School, Denver, Colorado 80220, U.S.A. (Received 29 June 1976) SUMMARY
1. The effect of the extracellular Ca concentration on the frequency of miniature end-plate potentials (min. e.p.p.s) at the frog neuromuscular junction was studied. 2. In saline containing elevated K (5 or 11 mM), the frequency of min. e.p.p.s increased as Ca concentration was increased from 0-1 to 1-3 mm. However, with further increases of Ca concentration up to 10 mM, min. e.p.p. frequency declined. 3. In saline containing the normal concentration of K (2 mM), increasing Ca concentration from 0- to 10 mm produced a slight, monotonic increase in mm. e.p.p. frequency. 4. The non-monotonic effect of Ca on min. e.p.p. frequency in preparations depolarized by elevated K is consistent with the existence of two opposing effects of Ca on transmitter release. Firstly, raising the external concentration of Ca increases the electrochemical potential for Ca entry, which tends to increase Ca influx and transmitter release. Secondly, increasing external Ca concentration increases electrostatic screening of fixed negative charges on the outer surface of the nerve terminal membrane. Such an increase in screening of charges near voltage-sensitive Ca gates would produce a hyperpolarization across the gates and they would tend to close, an effect which would tend to decrease Ca influx. The monotonic increase in min. e.p.p. frequency with increasing Ca concentration in 2 mM-K is consistent with the voltage insensitivity of the Ca gates at potentials close to the normal resting potential. INTRODUCTION
Calcium is known to be important in the linkage between depolarization of motor nerve terminals and the release of transmitter quanta at the neuromuscular junction (del Castillo & Katz, 1954; Katz & Miledi, 1965,
G. MATTHEWS AND W. 0. WICKELGREN 92 1967). Katz & Miledi (1969) obtained evidence that synaptic terminal membrane at the squid giant synapse contains Ca channels which open upon depolarization, allowing influx of calcium ions. Thus, depolarization leads to an increase in intracellular Ca concentration, which in some manner increases the release of transmitter quanta (see Rahamimoff, Erulkar, Alnaes, Meiri, Rotshenker & Rahamimoff, 1976). In addition to its effect on transmitter release, Ca also affects the excitability of nerve membranes. Frankenhaeuser & Hodgkin (1957) determined that reduction of Ca had an effect on the Hodgkin-Huxley parameters, m, n and h similar to that produced by a steady depolarization. There is now considerable evidence that this effect of Ca (and other divalent and trivalent cations as well) is due to its ability to screen the electrostatic effect of fixed negative charges which are located on the outer surface of nerve membranes near the voltage-sensitive gating mechanisms of ion channels (see Hille, Woodhull & Shapiro, 1975). Thus, when the concentration of Ca, [Ca], is reduced, the fixed negative charges are less well screened and the voltage-sensitive gating mechanisms of the membrane sense an effective depolarization which, however, is not recorded by an intracellular electrode. In the previous paper (Matthews & Wickelgren, 1977) we reported that guanidine (CH5N3) both increased neural excitability and potentiated transmitter release. We proposed that both of these effects could be explained by assuming that guanidine, a monovalent cation, binds to the outside of the nerve membrane in the regions of fixed negative charges around gated ion channels and reduces the electrostatic screening of these charges by divalent cations. The resulting effective depolarization would increase both neural excitability and transmitter release by tending to open gated Na, K, and Ca channels. This hypothesis for the membrane effects of guanidine suggests in turn that external Ca ions may normally have an effect not only on the electrochemical potential for Ca ('electrochemical potential' is used here and in the remainder of the paper in a very loose sense to denote the effect of [Ca] on both the Ca equilibrium potential and, for any given level of Ca permeability, Ca conductance) but also on the effective membrane potential across the gated channels. Consideration of these two separate effects of Ca leads to the prediction that manipulation of [Ca] would have two opposing effects on transmitter release. For example, reduction in [Ca] would: (1) reduce the electrochemical potential for Ca entry into nerve terminals, tending to diminish the influx of Ca into the nerve terminal and thereby reduce transmitter release; and (2) decrease the screening of negative charges near Ca channels thus producing an effective depolarization of the gating mechanism and increasing the number of open Ca channels. This second effect would tend
93 Ca EFFECT ON MIN. E.P.P.S to increase the influx of Ca and thereby increase transmitter release. In this view the effect of altering [Ca] on transmitter release would depend upon the quantitative balance between these two opposing effects on calcium influx. The effect of [Ca] on the membrane potential across gated Ca channels appears not to have been considered in previous studies on the role of Ca in transmitter release. It would be difficult to detect this effect on [Ca] on transmitter release evoked by action potentials, since the large depolarization produced by the action potential would tend to mask the small alterations in effective membrane potential produced by modest changes in [Ca]. Further, experimental manipulations of [Ca] can be expected to have effects on the size and shape of the action potential (Frankenhaeuser, 1957) and this would further complicate the analysis. Therefore, we decided to investigate the relationship between [Ca] and the rate of occurrence of spontaneous miniature end-plate potentials (min. e.p.p.s) at the frog neuromuscular junction. Previous observations on the effect of [Ca] on min. e.p.p. frequency in this preparation have reported that reducing [Ca] from 1 0 to 0 mm reduces min. e.p.p. frequency (Blioch, Glagoleva, Liberman & Nenashev, 1968) and raising [Ca] from 1P8 to 15 mM increases min. e.p.p. frequency (Rotshenker & Rahamimoff, 1970). These observations at frog neuromuscular junction are in accord with those made at the mammalian neuromuscular junction (Liley, 1956; Hubbard, 1961; Elmqvist & Feldman, 1965) and appear to reflect only an effect of [Ca] on the electrochemical potential for Ca entry into the nerve terminal. However, one would not expect to be able to detect an effect of [Ca] on the gating of Ca channels if the manipulations of [Ca] were made at a nerve terminal membrane potential around which modest changes in potential have little or no effect on the opening of Ca channels. Indeed, there is evidence that for frog motor nerve terminals a 5-10 mV depolarization, calculated from the Goldman-Hodgkin-Katz equation for a rise in extracellular [K] from 2 mm (normal saline) to approximately 4 mM, produces no increase in min. e.p.p. frequency (Takeuchi & Takeuchi, 1961; Matthews & Wickelgren, 1977). These observations indicate that the resting low Ca permeability of frog motor nerve terminals does not begin to rise until the membrane has been depolarized by more than 5-10 mV. However, if the nerve terminal membrane were depolarized sufficiently so that the voltagedependent opening of Ca channels became sensitive to small changes in membrane potential, then one might expect to see in the relationship between [Ca] and min. e.p.p. frequency effects which are attributable to the interactions of the opposing actions of Ca proposed here. Therefore, using the frog neuromuscular junction, we studied the relationship between min. e.p.p. frequency and [Ca] with the motor nerve terminals at different
94
G. MATTHEWS AND W. 0. WICKELGREN levels of steady membrane potential as set by adjusting the [K] of the saline. The results fit qualitatively with what might be expected from two opposing effects of Ca on spontaneous transmitter release and suggest that a complete description of the effect of Ca on transmitter release should include both effects. METHODS
Dissection of frog cutaneus pectoris muscle, composition of normal frog saline and recording conditions were described in the previous paper (Matthews & Wickelgren, 1977). For experiments in which it was desired to produce a steady depolarization of the motor nerve terminals the [K] of the saline was increased to either 5 or 11 mm. Test salines contained the particular [K] selected for that day's experiment and varying amounts of Ca (0-1-10 mM). For all test salines the [Na] was altered to maintain osmolarity equal to that of normal saline. Neostigmine methylsulphate (Roche) at 1 ,ug/ml. was added to all solutions. The normal procedure for data collection was to obtain for each end-plate a continuous record of min. e.p.p.s in normal saline and in two test salines. After changing solutions a minimum of 5 min was allowed to elapse before taking a record of min. e.p.p.s in order to permit stabilization of temperature. The effect of a test saline on min. e.p.p. frequency was expressed as the ratio of the mean min. e.p.p. frequency in the test saline to the mean min. e.p.p. frequency in normal saline (F/FO). This normalization procedure permitted combining data from different endplates with different mean min. e.p.p. frequencies in normal saline. This was the usual procedure for collecting data, and it allowed determination of the effects of various test salines on min. e.p.p. frequency at a number of end-plates without having to hold a particular end-plate through more than two changes of saline. However, in one experiment the same end-plate was held through five changes of saline as [Ca] was varied from 0-5-10 mm with a constant 11 mM-K. RESULTS
The relationship between [Ca] and min. e.p.p. frequency was nonmonotonic in solutions containing 5 or 11 mM-K. Increase in [Ca] up to 1-3 mm produces increases in min. e.p.p. frequency but further increases in Ca concentration caused the frequency of min. e.p.p.s to fall. This nonmonotonic effect of [Ca] is illustrated in Fig. 1, which shows sample records of min. e.p.p.s at three levels of external Ca at an end-plate bathed in saline containing 11 mM-K. When the concentration of Ca was increased from 0-5 to 1 0 mM, the average min. e.p.p. frequency increased from 19-5 + 4-6/ see to 43-1 + 3-9/sec (mean + S.D. of observation), as would be expected from the increase in electrochemical potential for Ca entry. However, a further increase of external Ca up to 6-0 mm resulted in a decrease in min. e.p.p. frequency to 12-1 + 4-4/sec which was below that observed in 0-5 mM-Ca. The complete results from the experiment on this end-plate are presented in Fig. 2. It is apparent from the figure that the decline in min. e.p.p. frequency observed in 6-0 mM-Ca is part of a progressive decrease as [Ca] was increased from 1 -0 to 10 mm. This effect is not expected
Ca EFFECT ON MIN. E.P.PS 95 from consideration of the effect of Ca concentration on electrochemical potential alone and is attributed to a hyperpolarizing effect on the Ca gates produced by increased [Ca]. In saline containing normal K concentration (2 mM), the results were qualitatively different from those just described. As [Ca] was raised from A
M\JJfrkkLJ&i
,LJ\
B
a C
Lx|Jj 2 mV 0-2 sec Fig. 1. Effect of external Ca concentration on frequency of miniature endplate potentials (min. e.p.p.s) at the frog neuromuscular junction: A, 0 5 mm-Ca; B, -0 mm-Ca; and 0, 6-0 mm-Ca. The extracellular K concentration was 11 mM in all cases. The photographs of strip-chart records shown at each level of Ca are continuous 4-8 sec samples. The highest frequency was observed in 1-0 mm-Ca, both 0-5 and 6-0 mM-Ca being lower. 4
P HY 266
G. MATTHEWS AND W. 0. WICKELGREN 0 5 to 10 mM, min. e.p.p. frequency increased slightly and no decrease at higher levels of [Ca] was observed. In Fig. 3, sample results from an endplate in normal saline show a monotonic effect of Ca concentration on min. e.p.p. frequency, in contrast to the effect observed in 11 mM-K. This small increase in average min. e.p.p. frequency with increasing [Ca] but normal [K] is consistent with previous studies of min.e.p.p. frequency at the frog neuromuscular junction (Blioch et al. 1968; Rotshenker & Rahamimoff, 1970). 96
50 45
40 35
U
Cr25 CL 20 4;
15 _ lA.
,
,
,
,
4
5
6
7
10 0
1
2
3
8
9
10
[Ca2+]O (mM) Fig. 2. Effect of external Ca concentration, [Ca2+]0, on average min. e.p.p. frequency at an end-plate bathed in saline containing 11 mm-K. The endplate is the same one from which the examples shown in Fig. 1 were taken. The points represent the mean of successive 1 sec counts during the 30 see observation period at each value of Ca concentration and the dashed lines and bars indicate + I S.D. of observation.
The examples presented above were of experiments on single end-plates but, in most experiments recordings were made from the same end-plate in only two test solutions in addition to normal saline and results from separate end-plates were normalized (see Methods and heading of Table 1). Results expressed in this way from three different preparations (one each in 2, 5 and 11 mM-K) are presented in Fig. 4. The results shown are qualitatively the same as the results from single end-plates presented in Figs. 2 and 3. In solutions containing the normal level of external K,
Ca EFFECT ON MIN. E.PYS 97 relative min. e.p.p. frequency increased steadily as [Ca] was raised from 0 5 to 6-0 mm, although there was no further increase in frequency as [Ca] was raised to 10 mm. At higher levels of K (5 and II mM), the relationship between Ca concentration and relative frequency was distinctly nonmonotonic. The normalized min. e.p.p. frequencies for nine experiments are 06 T
05 0-4
_
A
T
0-2
01 1
2
5 6 7 8 9 10 [Ca2+]o (mM) Fig. 3. Effect of external Ca concentration, [Ca2+]O, on average, min. e.p.p. 0
3
4
frequency at an end-plate bathed in saline containing normal K (2 mm). The points represent the mean of successive 40 sec counts during the 560 sec observation period at each value of Ca concentration and the dashed lines and bars indicate + 1 S.D. of observation.
shown in Table 1, from which it can be seen that the results shown in Fig. 4 were observed in all experiments, without exception. In each experiment in raised K, the miniature potential frequency reached a maximum at relatively low Ca concentrations (0.5 to 3 mM) and declined thereafter. DISCUSSION
The present results are consistent with the proposed dual effect of Ca on transmitter release. When the nerve terminals were depolarized by 5 or it mM-K, the relationship between min. e.p.p. frequency and [Ca] was distinctly non-monotonic. This effect can be understood in terms of our proposed model of the opposing effects of [Ca] on (1) the electrochemical potential for Ca entry and (2) the effective membrane potential across 4-2
G. MATTHEWS AND W. 0. WICKELGREN 98 gated Ca channels. The qualitative explanation is as follows. As [Ca] is increased, negative charges attached to the outside of the nerve terminal membrane are increasingly screened. The reduction of the electrostatic effect of these negative charges produces an effective hyperpolarization of the voltage-sensitive Ca-gating mechanisms. Thus, some Ca gates close and 100*0 500 20-0 -~~
200
i.0
2.0
-
I
:
IIII
1-0
0 5 -0
2
4
6
8
10
[Ca2+]0(mM)
Fig. 4. Frequency of mi. e.p.p.s at the frog neuromuscular junction as a function of extracellular Ca concentration: open circles, 11 mm-K; open triangles, 5 mM-K; open squares, 2 mM-K. The frequency on the ordinate is expressed as the ratio of the frequency in the particular test solution (F) to the frequency (Fo) in normal saline (2 mM-K, 2 mM-Ca). Each data point at a particular value of [Ca] represents the value of F/Fe at a different end-plate. The data for 2 mm are from muscle number (3) of Table 1, the data for 5 mM-K are from muscle number (4) of Table 1 and the data for 11 mm-K are from muscle number (7) of Table 1. Lines were drawn by eye.
the Ca permeability of the terminal membrane is decreased which, by itself, would decrease Ca influx and so decrease min. e.p.p. frequency. However, in the range of [Ca] from 0-1 mm to 1-3 mm, this decrease in the number of open Ca channels is more than offset by the increased electrochemical potential for Ca to enter through those channels which remain open and the result is an increase in min. e.p.p. frequency. However, with further increases in [Ca], the balance between the two opposing effects shifts. The gain in electrochemical potential is not sufficient to overcome the progressive decrease in Ca permeability, and the min. e.p.p. frequency decreases.
Ca EFFECT ON MIN. E.P.PS
99
TABLE 1. Effect of external Ca concentration, [Ca]0, on min. e.p.p. frequency at various levels of [K]0. Each number at a particular value of [Ca]o represents the value of F/FO at a single end-plate, where F/FO is the ratio of the mean min. e.p.p. frequency in the test saline to that of the min. e.p.p. frequency in normal saline (2.0 mM-K, 2-0 mM-Ca)
[K]o
Muscle number
2
1
[Ca]. 0-1 -
2 3 5
4
3-1
5
2-8 3-5 2-4
6
11
7 8
9
4-5 23-4
28*7 32-3
0-5
1.0
3-0
0-6 0-7 0-7 0-8
0-7
1-1
1-5 1-0 1-7 0-9
0-8 0-8 6-1 5-2 3-7 5-4 3-3 1-6 21-4 23-3 26-6 3-4
0-9
0-9
41-1 37-1 24-7
0-8
1-0 4-4 4-0 2-6 3-7 2-2 2-7 39-2
41-4 8-1 10-7 12-6 26-2 30-9 54-6
3-0 3-5 2-0 2-8 1-5 2-0 29-0 34-3 36-9 23-2 31-3 8-5 22-0 35-9
6-0 1-9
10-0 1-3
1-2 1-2
1-4 1-2 1-3 0-9 1-7 1-4 3-0 2-1 2-9 1-7 1-3 1-3 13-2 12-8
1-1 1-6 1-8 3-4 3-7 3-4 2-2 1-1 2-4 24-1 1-4 8-0
3-6 4-1
9-3 10-2 19-4 15-3
6-4 4-5
10-1 9-2
The inhibitory effect of Ca on min. e.p.p. frequency can be understood better by considering the relationship between min. e.p.p. frequency and nerve terminal membrane potential. At levels of depolarization induced by concentrations of K higher than 4 mM, min. e.p.p. frequency at the frog neuromuscular junction becomes sensitive to changes in membrane potential (see Fig. 5 of the previous paper, Matthews & Wickelgren, 1977). Thus, in the present experiments depolarization by exposure to 5 or 11 mM-K placed the terminals in the steep part of the depolarization-release curve. Therefore, small shifts in effective membrane potential resulting from slight changes of Ca would be expected to beimportant in influencing min.e.p.p. frequency and, indeed, appear to be more important than the concomitant, and opposing, changes in Ca electrochemical potential.
1. MATTHEWS AND W. 0. WICKELGREN In normal K (2 mM), the relationship between min. e.p.p. frequency and [Ca] was monotonic. This would appear to reflect the fact that at the frog neuromuscular junction min. e.p.p. frequency is insensitive to small changes in membrane potential around the normal resting potential, as evidenced by the flatness of the release curve between 2 and almost 4 mM-K (Takeuchi & Takeuchi, 1961; Matthews & Wickelgren, 1977). Thus in 2 mM-K, the effective hyperpolarization due to increasing [Ca] will have little effect on Ca permeability since few Ca channels are open. There remains only the 100
effect of [Ca] on Ca electrochemical potential, and this would account for the small monotonic effect of [Ca] on min. e.p.p. frequency. Gage & Quastel (1966) reported a non-monotonic effect of [Ca] on min. e.p.p. frequency at rat neuromuscular junctions depolarized by raising the concentration of K in the saline. Also, Cooke & Quastel (1973) observed that addition of Ca counteracted some of the effect of increasing [K] on min. e.p.p. frequency at the rat neuromuscular junction. In order to account for this inhibitory interaction between K and Ca, Cooke & Quastel suggested that there is a specific effect of K on min. e.p.p. frequency not mediated through an effect on membrane potential and that this specific effect of K is antagonized somehow by Ca. They did not consider the effect of Ca on effective membrane potential and from inspection of their published data it appears that their observations can also be explained by our model which has the advantage that it does not propose any new effects of either Ca or K. In our model the only assumption not based on established experimental findings is that there are fixed negative charges on the outer surface of the nerve membrane in the vicinity of voltagesensitive Ca gates and that Ca affects the screening of those charges in a manner similar to its effects on charges around Na and K channels. It is not clear what quantitative significance the effect of Ca on effective membrane potential has for studies on the role of Ca in transmitter release evoked by nerve action potentials. Frankenhaeuser & Hodgkin (1957) calculated a shift of 21-5 mV in the effective membrane potential across the Na channel in squid axon for every tenfold change in [Ca]. The magnitude of this shift in effective membrane potential depends upon the density of fixed charges around the gating mechanism but if there is a similar charge density around Ca channels, then the screening effect of Ca (and other divalent and trivalent cations) may be an important factor in evoked as well as spontaneous release and would need to be taken into account in a quantitative assessment of the effects of Ca on transmitter release. This work was supported by Research Grant NS 09661 and Research Career Development Award NS 50295. We wish to thank Mr George Tarver for help with the illustrations and Ms Carole Bucher for typing the manuscript.
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REFERENCES
BLIOCH, Z. L., GLAGOLEVA, I. M., LIBERMAN, E. A. & NENASHEV, V. A. (1968). A study of the mechanism of quantal transmitter release at a chemical synapse. J. Physiol. 199, 11-35. COOKE, J. D. & QUASTEL, D. M. J. (1973). The specific effect of potassium on transmitter release by rat motor nerve terminals, and its inhibition by calcium. J. Physiol. 228, 435-458. DEL CASTILLO, J. & KATZ, B. (1 954). Quantal components of the end-plate potential. J. Phyasol. 124, 560-573. ELMQVIST, D. & FELDMAN, D. S. (1965). Spontaneous activity at a mammalian neuromuscular junction in tetrodotoxin. Acta phyaiol. 8cand. 64, 475-476. FRANKENHAE USER, B. (1957). The effect of calcium on the myelinated nerve fibre. J. Physiol. 137, 245-260. FRANKENEAEUSER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical properties of squid axons. J. Phyaiol. 137, 218-244. GAGE, P. W. & QUASTEL, D. M. J. (1966). Competition between sodium and calcium ions in transmitter release at mammalian neuromuscular junctions. J. Phyaiol. 185, 95-123. HITLE, B., WOODHULL, A. M. & SHAPIRO, B. I. (1975). Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions and pH. Phil. Trans. R. Soc. B 270, 301-318. HUBBARD, J. I. (1961). The effect of calcium and magnesium on the spontaneous release of transmitter from mammalian motor nerve endings. J. Physiol. 159, 507-517. KATZ, B. & MILEDI, R. (1965). The effect of calcium on acetylcholine release from motor nerve endings. Proc. R. Soc. B 161, 496-503. KATZ, B. & MILEDI, R. (1967). A study of synaptic transmission in the absence of nerve impulses. J. Phyaiol. 192, 407-436. KATZ, B. & MILEDI, R. (1969). Tetrodotoxin-resistant electric activity in pre-synaptic terminals. J. Physiol. 203, 459-487. LILEY, A. W. (1956). The effects of presynaptic polarization on the spontaneous activity at the mammalian neuromuscular junction. J. Phyaiol. 134, 427-443. MATTHEwS, G. & WICKELGREN, W. 0. (1977). Effects of guanidine on transmitter release and neuronal excitability. J. Phygiol. 266, 69-89. RAHAMIMOFF, R., ERULKAR, S. D., ALNAEs, E., MEIRI, H., ROTSHENKER, S. & RAHAMIMOFF, H. (1976). Modulation of transmitter release by calcium ions and nerve impulses. Cold Spring Harb. Symp. quant. Biol. 40, 107-116. ROTSHENKER, S. & RAHAMIMOFF, R. (1970). Neuromuscular synapse: stochastic properties of spontaneous release of transmitter. Science, N.Y. 170, 648-649. TAEucmHI, A. & TAKEUCHI, N. (1961). Changes in potassium concentration around motor nerve terminals, produced by current flow, and their effects on neuromuscular transmission. J. Phy8iol. 155, 46-58.
91
ON THE EFFECT OF CALCIUM ON THE FREQUENCY OI MINIATURE END-PLATE POTENTIALS AT THE FROG NEUROMUSCULAR JUNCTION
BY GARY MATTHEWS AND WARREN 0. WICKELGREN From the Department of Physiology, University of Colorado Medical School, Denver, Colorado 80220, U.S.A. (Received 29 June 1976) SUMMARY
1. The effect of the extracellular Ca concentration on the frequency of miniature end-plate potentials (min. e.p.p.s) at the frog neuromuscular junction was studied. 2. In saline containing elevated K (5 or 11 mM), the frequency of min. e.p.p.s increased as Ca concentration was increased from 0-1 to 1-3 mm. However, with further increases of Ca concentration up to 10 mM, min. e.p.p. frequency declined. 3. In saline containing the normal concentration of K (2 mM), increasing Ca concentration from 0- to 10 mm produced a slight, monotonic increase in mm. e.p.p. frequency. 4. The non-monotonic effect of Ca on min. e.p.p. frequency in preparations depolarized by elevated K is consistent with the existence of two opposing effects of Ca on transmitter release. Firstly, raising the external concentration of Ca increases the electrochemical potential for Ca entry, which tends to increase Ca influx and transmitter release. Secondly, increasing external Ca concentration increases electrostatic screening of fixed negative charges on the outer surface of the nerve terminal membrane. Such an increase in screening of charges near voltage-sensitive Ca gates would produce a hyperpolarization across the gates and they would tend to close, an effect which would tend to decrease Ca influx. The monotonic increase in min. e.p.p. frequency with increasing Ca concentration in 2 mM-K is consistent with the voltage insensitivity of the Ca gates at potentials close to the normal resting potential. INTRODUCTION
Calcium is known to be important in the linkage between depolarization of motor nerve terminals and the release of transmitter quanta at the neuromuscular junction (del Castillo & Katz, 1954; Katz & Miledi, 1965,
G. MATTHEWS AND W. 0. WICKELGREN 92 1967). Katz & Miledi (1969) obtained evidence that synaptic terminal membrane at the squid giant synapse contains Ca channels which open upon depolarization, allowing influx of calcium ions. Thus, depolarization leads to an increase in intracellular Ca concentration, which in some manner increases the release of transmitter quanta (see Rahamimoff, Erulkar, Alnaes, Meiri, Rotshenker & Rahamimoff, 1976). In addition to its effect on transmitter release, Ca also affects the excitability of nerve membranes. Frankenhaeuser & Hodgkin (1957) determined that reduction of Ca had an effect on the Hodgkin-Huxley parameters, m, n and h similar to that produced by a steady depolarization. There is now considerable evidence that this effect of Ca (and other divalent and trivalent cations as well) is due to its ability to screen the electrostatic effect of fixed negative charges which are located on the outer surface of nerve membranes near the voltage-sensitive gating mechanisms of ion channels (see Hille, Woodhull & Shapiro, 1975). Thus, when the concentration of Ca, [Ca], is reduced, the fixed negative charges are less well screened and the voltage-sensitive gating mechanisms of the membrane sense an effective depolarization which, however, is not recorded by an intracellular electrode. In the previous paper (Matthews & Wickelgren, 1977) we reported that guanidine (CH5N3) both increased neural excitability and potentiated transmitter release. We proposed that both of these effects could be explained by assuming that guanidine, a monovalent cation, binds to the outside of the nerve membrane in the regions of fixed negative charges around gated ion channels and reduces the electrostatic screening of these charges by divalent cations. The resulting effective depolarization would increase both neural excitability and transmitter release by tending to open gated Na, K, and Ca channels. This hypothesis for the membrane effects of guanidine suggests in turn that external Ca ions may normally have an effect not only on the electrochemical potential for Ca ('electrochemical potential' is used here and in the remainder of the paper in a very loose sense to denote the effect of [Ca] on both the Ca equilibrium potential and, for any given level of Ca permeability, Ca conductance) but also on the effective membrane potential across the gated channels. Consideration of these two separate effects of Ca leads to the prediction that manipulation of [Ca] would have two opposing effects on transmitter release. For example, reduction in [Ca] would: (1) reduce the electrochemical potential for Ca entry into nerve terminals, tending to diminish the influx of Ca into the nerve terminal and thereby reduce transmitter release; and (2) decrease the screening of negative charges near Ca channels thus producing an effective depolarization of the gating mechanism and increasing the number of open Ca channels. This second effect would tend
93 Ca EFFECT ON MIN. E.P.P.S to increase the influx of Ca and thereby increase transmitter release. In this view the effect of altering [Ca] on transmitter release would depend upon the quantitative balance between these two opposing effects on calcium influx. The effect of [Ca] on the membrane potential across gated Ca channels appears not to have been considered in previous studies on the role of Ca in transmitter release. It would be difficult to detect this effect on [Ca] on transmitter release evoked by action potentials, since the large depolarization produced by the action potential would tend to mask the small alterations in effective membrane potential produced by modest changes in [Ca]. Further, experimental manipulations of [Ca] can be expected to have effects on the size and shape of the action potential (Frankenhaeuser, 1957) and this would further complicate the analysis. Therefore, we decided to investigate the relationship between [Ca] and the rate of occurrence of spontaneous miniature end-plate potentials (min. e.p.p.s) at the frog neuromuscular junction. Previous observations on the effect of [Ca] on min. e.p.p. frequency in this preparation have reported that reducing [Ca] from 1 0 to 0 mm reduces min. e.p.p. frequency (Blioch, Glagoleva, Liberman & Nenashev, 1968) and raising [Ca] from 1P8 to 15 mM increases min. e.p.p. frequency (Rotshenker & Rahamimoff, 1970). These observations at frog neuromuscular junction are in accord with those made at the mammalian neuromuscular junction (Liley, 1956; Hubbard, 1961; Elmqvist & Feldman, 1965) and appear to reflect only an effect of [Ca] on the electrochemical potential for Ca entry into the nerve terminal. However, one would not expect to be able to detect an effect of [Ca] on the gating of Ca channels if the manipulations of [Ca] were made at a nerve terminal membrane potential around which modest changes in potential have little or no effect on the opening of Ca channels. Indeed, there is evidence that for frog motor nerve terminals a 5-10 mV depolarization, calculated from the Goldman-Hodgkin-Katz equation for a rise in extracellular [K] from 2 mm (normal saline) to approximately 4 mM, produces no increase in min. e.p.p. frequency (Takeuchi & Takeuchi, 1961; Matthews & Wickelgren, 1977). These observations indicate that the resting low Ca permeability of frog motor nerve terminals does not begin to rise until the membrane has been depolarized by more than 5-10 mV. However, if the nerve terminal membrane were depolarized sufficiently so that the voltagedependent opening of Ca channels became sensitive to small changes in membrane potential, then one might expect to see in the relationship between [Ca] and min. e.p.p. frequency effects which are attributable to the interactions of the opposing actions of Ca proposed here. Therefore, using the frog neuromuscular junction, we studied the relationship between min. e.p.p. frequency and [Ca] with the motor nerve terminals at different
94
G. MATTHEWS AND W. 0. WICKELGREN levels of steady membrane potential as set by adjusting the [K] of the saline. The results fit qualitatively with what might be expected from two opposing effects of Ca on spontaneous transmitter release and suggest that a complete description of the effect of Ca on transmitter release should include both effects. METHODS
Dissection of frog cutaneus pectoris muscle, composition of normal frog saline and recording conditions were described in the previous paper (Matthews & Wickelgren, 1977). For experiments in which it was desired to produce a steady depolarization of the motor nerve terminals the [K] of the saline was increased to either 5 or 11 mm. Test salines contained the particular [K] selected for that day's experiment and varying amounts of Ca (0-1-10 mM). For all test salines the [Na] was altered to maintain osmolarity equal to that of normal saline. Neostigmine methylsulphate (Roche) at 1 ,ug/ml. was added to all solutions. The normal procedure for data collection was to obtain for each end-plate a continuous record of min. e.p.p.s in normal saline and in two test salines. After changing solutions a minimum of 5 min was allowed to elapse before taking a record of min. e.p.p.s in order to permit stabilization of temperature. The effect of a test saline on min. e.p.p. frequency was expressed as the ratio of the mean min. e.p.p. frequency in the test saline to the mean min. e.p.p. frequency in normal saline (F/FO). This normalization procedure permitted combining data from different endplates with different mean min. e.p.p. frequencies in normal saline. This was the usual procedure for collecting data, and it allowed determination of the effects of various test salines on min. e.p.p. frequency at a number of end-plates without having to hold a particular end-plate through more than two changes of saline. However, in one experiment the same end-plate was held through five changes of saline as [Ca] was varied from 0-5-10 mm with a constant 11 mM-K. RESULTS
The relationship between [Ca] and min. e.p.p. frequency was nonmonotonic in solutions containing 5 or 11 mM-K. Increase in [Ca] up to 1-3 mm produces increases in min. e.p.p. frequency but further increases in Ca concentration caused the frequency of min. e.p.p.s to fall. This nonmonotonic effect of [Ca] is illustrated in Fig. 1, which shows sample records of min. e.p.p.s at three levels of external Ca at an end-plate bathed in saline containing 11 mM-K. When the concentration of Ca was increased from 0-5 to 1 0 mM, the average min. e.p.p. frequency increased from 19-5 + 4-6/ see to 43-1 + 3-9/sec (mean + S.D. of observation), as would be expected from the increase in electrochemical potential for Ca entry. However, a further increase of external Ca up to 6-0 mm resulted in a decrease in min. e.p.p. frequency to 12-1 + 4-4/sec which was below that observed in 0-5 mM-Ca. The complete results from the experiment on this end-plate are presented in Fig. 2. It is apparent from the figure that the decline in min. e.p.p. frequency observed in 6-0 mM-Ca is part of a progressive decrease as [Ca] was increased from 1 -0 to 10 mm. This effect is not expected
Ca EFFECT ON MIN. E.P.PS 95 from consideration of the effect of Ca concentration on electrochemical potential alone and is attributed to a hyperpolarizing effect on the Ca gates produced by increased [Ca]. In saline containing normal K concentration (2 mM), the results were qualitatively different from those just described. As [Ca] was raised from A
M\JJfrkkLJ&i
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B
a C
Lx|Jj 2 mV 0-2 sec Fig. 1. Effect of external Ca concentration on frequency of miniature endplate potentials (min. e.p.p.s) at the frog neuromuscular junction: A, 0 5 mm-Ca; B, -0 mm-Ca; and 0, 6-0 mm-Ca. The extracellular K concentration was 11 mM in all cases. The photographs of strip-chart records shown at each level of Ca are continuous 4-8 sec samples. The highest frequency was observed in 1-0 mm-Ca, both 0-5 and 6-0 mM-Ca being lower. 4
P HY 266
G. MATTHEWS AND W. 0. WICKELGREN 0 5 to 10 mM, min. e.p.p. frequency increased slightly and no decrease at higher levels of [Ca] was observed. In Fig. 3, sample results from an endplate in normal saline show a monotonic effect of Ca concentration on min. e.p.p. frequency, in contrast to the effect observed in 11 mM-K. This small increase in average min. e.p.p. frequency with increasing [Ca] but normal [K] is consistent with previous studies of min.e.p.p. frequency at the frog neuromuscular junction (Blioch et al. 1968; Rotshenker & Rahamimoff, 1970). 96
50 45
40 35
U
Cr25 CL 20 4;
15 _ lA.
,
,
,
,
4
5
6
7
10 0
1
2
3
8
9
10
[Ca2+]O (mM) Fig. 2. Effect of external Ca concentration, [Ca2+]0, on average min. e.p.p. frequency at an end-plate bathed in saline containing 11 mm-K. The endplate is the same one from which the examples shown in Fig. 1 were taken. The points represent the mean of successive 1 sec counts during the 30 see observation period at each value of Ca concentration and the dashed lines and bars indicate + I S.D. of observation.
The examples presented above were of experiments on single end-plates but, in most experiments recordings were made from the same end-plate in only two test solutions in addition to normal saline and results from separate end-plates were normalized (see Methods and heading of Table 1). Results expressed in this way from three different preparations (one each in 2, 5 and 11 mM-K) are presented in Fig. 4. The results shown are qualitatively the same as the results from single end-plates presented in Figs. 2 and 3. In solutions containing the normal level of external K,
Ca EFFECT ON MIN. E.PYS 97 relative min. e.p.p. frequency increased steadily as [Ca] was raised from 0 5 to 6-0 mm, although there was no further increase in frequency as [Ca] was raised to 10 mm. At higher levels of K (5 and II mM), the relationship between Ca concentration and relative frequency was distinctly nonmonotonic. The normalized min. e.p.p. frequencies for nine experiments are 06 T
05 0-4
_
A
T
0-2
01 1
2
5 6 7 8 9 10 [Ca2+]o (mM) Fig. 3. Effect of external Ca concentration, [Ca2+]O, on average, min. e.p.p. 0
3
4
frequency at an end-plate bathed in saline containing normal K (2 mm). The points represent the mean of successive 40 sec counts during the 560 sec observation period at each value of Ca concentration and the dashed lines and bars indicate + 1 S.D. of observation.
shown in Table 1, from which it can be seen that the results shown in Fig. 4 were observed in all experiments, without exception. In each experiment in raised K, the miniature potential frequency reached a maximum at relatively low Ca concentrations (0.5 to 3 mM) and declined thereafter. DISCUSSION
The present results are consistent with the proposed dual effect of Ca on transmitter release. When the nerve terminals were depolarized by 5 or it mM-K, the relationship between min. e.p.p. frequency and [Ca] was distinctly non-monotonic. This effect can be understood in terms of our proposed model of the opposing effects of [Ca] on (1) the electrochemical potential for Ca entry and (2) the effective membrane potential across 4-2
G. MATTHEWS AND W. 0. WICKELGREN 98 gated Ca channels. The qualitative explanation is as follows. As [Ca] is increased, negative charges attached to the outside of the nerve terminal membrane are increasingly screened. The reduction of the electrostatic effect of these negative charges produces an effective hyperpolarization of the voltage-sensitive Ca-gating mechanisms. Thus, some Ca gates close and 100*0 500 20-0 -~~
200
i.0
2.0
-
I
:
IIII
1-0
0 5 -0
2
4
6
8
10
[Ca2+]0(mM)
Fig. 4. Frequency of mi. e.p.p.s at the frog neuromuscular junction as a function of extracellular Ca concentration: open circles, 11 mm-K; open triangles, 5 mM-K; open squares, 2 mM-K. The frequency on the ordinate is expressed as the ratio of the frequency in the particular test solution (F) to the frequency (Fo) in normal saline (2 mM-K, 2 mM-Ca). Each data point at a particular value of [Ca] represents the value of F/Fe at a different end-plate. The data for 2 mm are from muscle number (3) of Table 1, the data for 5 mM-K are from muscle number (4) of Table 1 and the data for 11 mm-K are from muscle number (7) of Table 1. Lines were drawn by eye.
the Ca permeability of the terminal membrane is decreased which, by itself, would decrease Ca influx and so decrease min. e.p.p. frequency. However, in the range of [Ca] from 0-1 mm to 1-3 mm, this decrease in the number of open Ca channels is more than offset by the increased electrochemical potential for Ca to enter through those channels which remain open and the result is an increase in min. e.p.p. frequency. However, with further increases in [Ca], the balance between the two opposing effects shifts. The gain in electrochemical potential is not sufficient to overcome the progressive decrease in Ca permeability, and the min. e.p.p. frequency decreases.
Ca EFFECT ON MIN. E.P.PS
99
TABLE 1. Effect of external Ca concentration, [Ca]0, on min. e.p.p. frequency at various levels of [K]0. Each number at a particular value of [Ca]o represents the value of F/FO at a single end-plate, where F/FO is the ratio of the mean min. e.p.p. frequency in the test saline to that of the min. e.p.p. frequency in normal saline (2.0 mM-K, 2-0 mM-Ca)
[K]o
Muscle number
2
1
[Ca]. 0-1 -
2 3 5
4
3-1
5
2-8 3-5 2-4
6
11
7 8
9
4-5 23-4
28*7 32-3
0-5
1.0
3-0
0-6 0-7 0-7 0-8
0-7
1-1
1-5 1-0 1-7 0-9
0-8 0-8 6-1 5-2 3-7 5-4 3-3 1-6 21-4 23-3 26-6 3-4
0-9
0-9
41-1 37-1 24-7
0-8
1-0 4-4 4-0 2-6 3-7 2-2 2-7 39-2
41-4 8-1 10-7 12-6 26-2 30-9 54-6
3-0 3-5 2-0 2-8 1-5 2-0 29-0 34-3 36-9 23-2 31-3 8-5 22-0 35-9
6-0 1-9
10-0 1-3
1-2 1-2
1-4 1-2 1-3 0-9 1-7 1-4 3-0 2-1 2-9 1-7 1-3 1-3 13-2 12-8
1-1 1-6 1-8 3-4 3-7 3-4 2-2 1-1 2-4 24-1 1-4 8-0
3-6 4-1
9-3 10-2 19-4 15-3
6-4 4-5
10-1 9-2
The inhibitory effect of Ca on min. e.p.p. frequency can be understood better by considering the relationship between min. e.p.p. frequency and nerve terminal membrane potential. At levels of depolarization induced by concentrations of K higher than 4 mM, min. e.p.p. frequency at the frog neuromuscular junction becomes sensitive to changes in membrane potential (see Fig. 5 of the previous paper, Matthews & Wickelgren, 1977). Thus, in the present experiments depolarization by exposure to 5 or 11 mM-K placed the terminals in the steep part of the depolarization-release curve. Therefore, small shifts in effective membrane potential resulting from slight changes of Ca would be expected to beimportant in influencing min.e.p.p. frequency and, indeed, appear to be more important than the concomitant, and opposing, changes in Ca electrochemical potential.
1. MATTHEWS AND W. 0. WICKELGREN In normal K (2 mM), the relationship between min. e.p.p. frequency and [Ca] was monotonic. This would appear to reflect the fact that at the frog neuromuscular junction min. e.p.p. frequency is insensitive to small changes in membrane potential around the normal resting potential, as evidenced by the flatness of the release curve between 2 and almost 4 mM-K (Takeuchi & Takeuchi, 1961; Matthews & Wickelgren, 1977). Thus in 2 mM-K, the effective hyperpolarization due to increasing [Ca] will have little effect on Ca permeability since few Ca channels are open. There remains only the 100
effect of [Ca] on Ca electrochemical potential, and this would account for the small monotonic effect of [Ca] on min. e.p.p. frequency. Gage & Quastel (1966) reported a non-monotonic effect of [Ca] on min. e.p.p. frequency at rat neuromuscular junctions depolarized by raising the concentration of K in the saline. Also, Cooke & Quastel (1973) observed that addition of Ca counteracted some of the effect of increasing [K] on min. e.p.p. frequency at the rat neuromuscular junction. In order to account for this inhibitory interaction between K and Ca, Cooke & Quastel suggested that there is a specific effect of K on min. e.p.p. frequency not mediated through an effect on membrane potential and that this specific effect of K is antagonized somehow by Ca. They did not consider the effect of Ca on effective membrane potential and from inspection of their published data it appears that their observations can also be explained by our model which has the advantage that it does not propose any new effects of either Ca or K. In our model the only assumption not based on established experimental findings is that there are fixed negative charges on the outer surface of the nerve membrane in the vicinity of voltagesensitive Ca gates and that Ca affects the screening of those charges in a manner similar to its effects on charges around Na and K channels. It is not clear what quantitative significance the effect of Ca on effective membrane potential has for studies on the role of Ca in transmitter release evoked by nerve action potentials. Frankenhaeuser & Hodgkin (1957) calculated a shift of 21-5 mV in the effective membrane potential across the Na channel in squid axon for every tenfold change in [Ca]. The magnitude of this shift in effective membrane potential depends upon the density of fixed charges around the gating mechanism but if there is a similar charge density around Ca channels, then the screening effect of Ca (and other divalent and trivalent cations) may be an important factor in evoked as well as spontaneous release and would need to be taken into account in a quantitative assessment of the effects of Ca on transmitter release. This work was supported by Research Grant NS 09661 and Research Career Development Award NS 50295. We wish to thank Mr George Tarver for help with the illustrations and Ms Carole Bucher for typing the manuscript.
Ca EFFECT ON MIN. E.PPS
101
REFERENCES
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